ABSTRACT: PROJECT 3 One of the goals of the National Institute of Environmental Health Sciences Superfund Research Program (SRP) is the development of methods and technologies to detect, assess and evaluate the effects of toxic substances on human health. To address the issue of high analysis costs for assessing toxic substances in the environment as well as the need for measuring indicators of exposure to humans, this project will develop new immunoassays, improve the utility of the assays, and apply them (in collaboration with other investigators in the UC Davis SRP Center). A complement to gas or liquid chromatography (GC or LC) coupled to mass spectrometry (MS), immunoassays are characterized by their speed, sensitivity, high throughput and low cost, which is underscored by their long history of use in clinical diagnostics. This project proposes three aims: Aim I is to make strategic improvements to an emerging antibody technology (called VHH or nanobody), which will vastly change the field of immunodiagnostics, including testing for small environmental molecules. Aim II is to develop new assays for compounds of concern to the community partner, identified by the Bioremediation project (Project 1), and the SRP that can be used to assess hazardous chemicals in the environment and in humans. Some of the high-priority compounds for assay development include coumarin rodenticides, polychlorinated biphenyls (PCBs), and pesticides and their degradation products. In this same realm of antibody development, the second aim includes a collaboration with Projects 4 and 5 to develop antibodies for protein targets needed to enhance the throughput of Western blot technology. Western blots are critical components of diagnostic testing to identify the effects of toxic chemical exposure. With improved efficiency, more chemicals can be screened and a better grasp of the toxic effects can be discerned. Aim III is to advance field methods for chemical detection by developing new immunodiagnostic technologies and applying them to real-world sampling needs. Through a collaboration with the Lab-on-a-Chip Nanosensing Platforms project (Project 2), classical antibodies as well as newly developed VHH reagents will be used in novel biosensors and membrane-based platforms. These new technologies will emphasize field portability, high sample throughput, and ease of use by the end-user, in order augment the Program's research translation and community engagement efforts. Such technologies will be used to empower communities to collect data in response to their environmental chemical concerns. Ultimately, the immunoassays developed in this project will be valuable tools for stakeholders who wish to detect chemical exposures and biomarkers of their toxicity.